The timely and precise replication of the genome is critical to the normal proliferation of eukaryotic cells. Accordingly, the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long term objective of this proposal is to identify the proteins and mechanisms that direct the initiation of DNA replication at Saccharomyces cerevisiae chromosomes and to understand how these events are regulated during the cell cycle. A complex of six proteins, the origin recognition complex (ORC), binds a conserved sequence found in all S. cerevisiae chromosomal origin of DNA replication. This binding event requires ATP, indicating an important role for ATP in regulating ORC function. Cloning of the genes encoding each of the six subunits has provided genetic evidence consistent with this protein complex performing a central function in the initiation of DNA replication at yeast origins. To determine the role of ORC in yeast DNA replication the following specific experiments will be performed: The subunits responsible for DNA and ATP binding will be determined using a combination of ATP and DNA crosslinking and analysis of ORC assemblies missing one or more subunits. The regulation of ORC function by ATP will be investigated by studying the ATP binding and hydrolysis activities of ORC. The mechanisms coupling DNA and ATP binding will be determined. The proteins that interact with ORC will be sought using by protein affinity chromatography and by genetic screens for genes encoding such proteins. The connection between ORC and known replication and cell cycle proteins will be determined. Understanding the mechanisms directing yeast DNA replication will impact human health at two levels. First, because recent work on the eukaryotic cell cycle suggests that the mechanisms of DNA replication and its control are conserved among eukaryotes, findings in yeast will serve to direct future studies of DNA replication in human cells. Second, an understanding DNA replication and its regulation in yeast will provide new targets for the development of anti-fungal drugs and therapies.